Tim Söderlund

Comparison of the effects of clozapine, chlorpromazine, and haloperidol on membrane lateral heterogeneity

The interactions of three neuroleptic drugs, clozapine (CLZ), chlorpromazine (CPZ), and haloperidol (HPD) with phospholipids were compared using DSC and Langmuir balance. Main emphasis was on the drug-induced effects on the lateral organization of lipid mixtures of the saturated zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and the unsaturated acidic phosphatidylserine, brainPS. In multilamellar vesicles (MLV) phase separation was observed by DSC at X(PS)> or =0.05. All three drugs bound to these MLVs, abolishing the pretransition at X(drug)> or =0.03. The main transition temperature (T(m)) decreased almost linearly with increasing contents of the drugs, CLZ having the smallest effect. In distinction from the other two drugs, CLZ abolished the phase separation evident in the endotherms for DPPC/brainPS (X(PS)=0.05) MLVs. Compression isotherms of DPPC/brainPS/drug (X(PS)=X(drug)=0.05) monolayers revealed the neuroleptics to increase the average area/molecule, CLZ being the most effective. Penetration into brainPS monolayers showed strong interactions between the three drugs and this acidic phospholipid (in decreasing order CPZ>HPD>CLZ). Hydrophobic interactions demonstrated using neutral eggPC monolayers decreased in a different order, CLZ>CPZ>HPD. Fluorescence microscopy revealed domain morphology of DPPC/brainPS monolayers to be modulated by these drugs, increasing the gel-fluid domain boundary length in the phase coexistence region. To conclude, our data support the view that membrane-partitioning drugs could exert part of their effects by changing the lateral organization and thus also the functions of biomembranes.

Comparison of the Effects of Surface Tension and Osmotic Pressure on the Interfacial Hydration of a Fluid Phospholipid Bilayer

The effects of three so-called kosmotropic solutes, namely, betaine, sucrose, and choline chloride on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine large unilamellar vesicles, were studied by measuring the generalized polarization (GP) for the fluorescence emission of the membrane partitioning probe Laurdan. The latter has been shown to be sensitive to the depth of water penetration into phospholipid bilayers. At equal osmotic pressures the three solutes produced different increments in GP, with a qualitative positive correlation. However, the increments in GP correlated also quantitatively with the increase of air-water surface tension caused by the three kosmotropes. Our findings suggest surface tension to determine the impact of these solutes on the lateral packing of the lipid bilayer. Based on the changes in area/lipid at different surface tensions, the equilibrium lateral pressure for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer at 25 degrees C was estimated to be approximately 34 mN/m.

Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes.

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value.

Binding of Adriamycin to Liposomes as a Probe for Membrane Lateral Organization

A stopped-flow spectrofluorometer equipped with a rapid scanning emission monochromator was utilized to monitor the binding of adriamycin to phospholipid liposomes. The latter process is evident as a decrease in fluorescence emission from a trace amount of a pyrene-labeled phospholipid analog (PPDPG, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-++ +glyce rol) used as a donor for resonance energy transfer to adriamycin. For zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, fluorescence decay was slow, with a half-time t1/2 of approximately 2 s. When the mole fraction of the acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), was increased to XPG >/= 0.04, the decay of fluorescence became double exponential, and an additional, significantly faster process with t1/2 in the range between 2 and 4 ms was observed. Subsequently, as XPG was increased further, the amplitude of the fast process increased, whereas the slower process was attenuated, its t1/2 increasing to 20 s. Increasing [NaCl] above 50 mM or [CaCl2] above 150 microM abolished the fast component, thus confirming this interaction to be electrostatic. The critical dependence of the fast component on XPG allows the use of this process to probe the organization of acidic phospholipids in liposomes. This was demonstrated with 1, 2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes incorporating PPDPG (XPPDPG = 0.03), i.e., conditions where XPG in fluid bilayers is below the required threshold yielding the fast component. In keeping with the presence of clusters of PPDPG, the fast component was observed for gel-state liposomes. At approximately 34 degreesC (i.e., 6 degrees below Tm), the slower fluorescence decay also appeared, and it was seen throughout the main phase transition region as well as in the liquid-crystalline state. The fluorescence decay behavior at temperatures below, above, and at the main phase transition temperature is interpreted in terms of thermal density fluctuations and an intermediate state between gel and liquid-crystalline states being involved in the phospholipid main phase transition. This is the first observation of a cluster constituted by acidic phospholipids controlling the membrane association of a drug.

Interactions of Adriamycin, Cytochrome c, and Serum Albumin withLipid Monolayers Containing Poly(ethylene glycol)-Ceramide

Poly(ethylene glycol)(2000)C(20)ceramide (PEG-Cer) containing monolayers at an air/water interface were characterized by measuring their surface pressure versus area/molecule (pi-A) and surface potential versus area/molecule (Delta V-A) isotherms. The behavior of pi-A as well as Delta V versus lipid density (Delta V-n) and Delta V-pi isotherms for PEG-Cer are in keeping with two transitions of the lipopolymer, starting at pi approximately equal to 9 and 21 mN/m. We also investigated the effects of PEG-Cer on the binding of adriamycin, cytochrome c and bovine serum albumin to monolayers containing varying mole fractions X of PEG-Cer. PEG-Cer impedes the penetration of these ligands into lipid monolayers with similar effects at both X = 0.04 and 0.08. This effect of PEG-Cer depends on the conformation of the lipopolymer and the interactions between the lipid surface and the surface-interacting molecule as well as the size of the latter.

Effects of betaine on the structural dynamics of Thermomyces (Humicola) lanuginosa lipase

Abstract The impact of betaine on the structural dynamics of a soluble protein, Thermomyces (Humicola) lanuginosa lipase (HLL) was studied using differential scanning calorimetry (DSC), surface tension measurements, and both steady-state and time-resolved fluorescence spectroscopy. Betaine increased the thermal stability of wild type HLL and protected this enzyme from unfolding by the chaotrope, guanidine hydrochloride. Time-resolved fluorescence anisotropy measurements revealed betaine to decrease the mean hydrodynamic volume of the single Trp HLL mutant, W89m, and to reduce the movements of the α-helical segment constituting the so-called ‘lid’. In the presence of betaine, the average environment of Trp89 became more hydrophobic with decreased accessibility to this residue for the water soluble quencher I−, the bimolecular collisional rate constant, kq decreasing from 6.86×108 to 5.06×108 M−1 s−1 in 1.0 M betaine. The above results are in keeping with an augmented packing of the lipase in the presence of betaine. Betaine increases surface tension (γ) of water and enhances the denaturation of HLL into the air–water interface. The increased stability of HLL and increased γ by betaine suggest low or no binding of betaine to the surface of protein. The effects of betaine on the structural dynamics of HLL are readily explained by the increased surface tension of water caused by this solute.

The role of outpatient visit after operative treatment of ankle fractures

It is a common practice that patients have a scheduled follow-up visit with radiographs following ankle fracture surgery. The aim of this study was to evaluate whether an early outpatient visit (<3 weeks) after ankle fracture surgery resulted in a change in patient management. For this study, 878 consecutive operatively treated ankle fracture patients with an early outpatient clinical-radiological visit were reviewed. The outcome measure was a change in treatment plan defined as any procedure, medication, or surgical intervention that is not typically implemented during the uncomplicated healing process of an acute fracture. A change in treatment plan was observed in 9.8% of operatively treated ankle fracture patients. The mean age of the patients was 48 years and the mean follow-up time was 64 months. Of the changes in treatment plan, 91% were exclusively due to clinical findings such as infection. Only three of 878 patients required a change in their treatment plan based merely on the findings of the radiographs taken at the outpatient visit. Only 37% of the patients requiring a change in their postoperative management had solicited an unanticipated visit before the scheduled outpatient visit due to clinical problems such as infection or a cast-related issue. Our study showed that every tenth operatively treated ankle fracture patient requires a change in their treatment plan due to a clinical problem such as infection or a cast-related issue. Although at hospital discharge all patients are provided with written instructions on where to contact if problems related to the operated ankle emerge, only one third of the patients are aware of the clinically alarming symptoms and seek care when problems present. Our findings do not support obtaining routine radiographs at the early outpatient visit in an ankle fracture patient without clinical signs of a complication.

Formation of a macroscopic membrane structure by liposomes containing cationic lipids and exposed to DC electric fields

Abstract Application of DC electric fields (from 20–30.4 kV/m) caused a rapid (within 1–5 s) formation of a large membrane structure in an aqueous solution of unilamellar liposomes composed of egg phosphatidylcholine and a cationic amphiphile, either sphingosine or stearylamine (mole fraction X=0.25). The formation of this structure could be readily observed by phase contrast microscopy. Maintenance of the membrane required the DC field and it disappeared rapidly (within ≈5 s) upon the removal of the field. The formation of the assembly was reversible. Such structures were not formed by vesicles composed of phosphatidylcholine only or containing instead of the cationic amphiphile an anionic lipid, phosphatidic acid. Fusion or a hemifusion of the vesicles is likely to take place as fluorescence microscopy of liposomes labeled with a pyrene-containing phospholipid analog revealed domain formation within the field-induced membrane, evident as a depletion of fluorescence in the region subjected to highest field intensity.

Resource use and clinical outcomes in blunt thoracic injury: a 10-year trauma registry comparison between southern Finland and Germany

PurposeSerious thoracic injuries are associated with high mortality, morbidity, and costs. We compared patient populations, treatment, and survival of serious thoracic injuries in southern Finland and Germany.MethodsMortality, patient characteristics and treatment modalities were compared over time (2006–2015) in all patients with Abbreviated Injury Scale (AIS) thorax ≥ 3, Injury Severity Score (ISS) > 15, age > 15 years, blunt trauma mechanism, and treatment in Intensive Care Unit (ICU) in Level 1 hospitals included in the Helsinki Trauma Registry (HTR) and the TraumaRegister DGU® (TR-DGU).ResultsWe included 934 patients from HTR and 25 448 patients from TR-DGU. Pre-hospital differences were seen between HTR and TR-DGU; transportation in the presence of a physician in 61% vs. 97%, helicopter use in 2% vs. 42%, intubation in 31% vs. 55%, and thoracostomy in 6% vs. 10% of cases, respectively. The mean hospital length of stay (LOS) and ICU LOS was shorter in HTR vs. TR-DGU (13 vs. 25 days and 9 vs. 12 days, respectively). Our main outcome measure, standardised mortality ratio, was not statistically significantly different [1.01, 95% confidence interval (CI) 0.84–1.18; HTR and 0.97, 95% CI 0.94–1.00; TR-DGU].ConclusionsMajor differences were seen in pre-hospital resources and use of pre-hospital intubation and thoracostomy. In Germany, pre-hospital intubation, tube thoracostomy, and on-scene physicians were more prevalent, while patients stayed longer in ICU and in hospital compared to Finland. Despite these differences in resources and treatment modalities, the standardised mortality of these patients was not statistically different.